Substituted porphyrins

ABSTRACT

Substituted metalloporphyrin compounds are described, along with pharmaceutical compositions containing the same, and methods of use thereof in protecting cells from oxidant-induced toxicity and pathological conditions such as inflammatory lung disease, neurodegenerative conditions, radiation injury, cancer, diabetes, cardiac conditions and sickle cell disease. Mn(III) porphyrins bearing oxygen atoms within side chains are particularly described.

TECHNICAL FIELD

The present invention relates generally to porphyrins and, moreparticularly to substituted porphyrins and to methods of using same.

BACKGROUND

Mn(III) cationic N-alkylpyridylporphyrin mimics of SOD activity havebeen developed (Spasojevic et al, J. Biol. Chem. 278:6831-6837 (2003),Batinic-Haberle, Methods Enzymol. 349:223-233 (2001), Spasojevic andBatinic-Haberle, Inorg. Chim. Acta. 317:230-242 (2001), Batinic-Haberleet al, J. Biol. Chem. 273:24521-24528 (1998), Batinic-Haberle et al,Inorg. Chem. 38:4011-4022 (1999), Kachadourian et al, Inorg. Chem.38:391-396 (1999), Batinic-Haberle et al, J. Chem. Soc., Dalton Trans.,pgs. 2689-2696 (2002), Ferrer-Sueta et al, J. Biol. Chem.278:27432-27438 (2003)) the N-ethylpyridyl derivative of which,MnTE-2-PyP⁵⁺ (Spasojevic et al, J. Biol. Chem. 278:6831-6837 (2003)),exhibits beneficial antioxidant properties in several animal models ofoxidative stress injury (Tao et al, Circulation 108:2805-2811 (2003),Sheng et al, J. Neurotrauma, In press (2003), Sheng et al, Drug News andPerspectives 15:654-665 (2002), Sheng et al, Free Radic. Biol. Med.33:947-961 (2002), Vujaskovic et al, Free Radic. Biol. Med. 33:857-863(2002), Piganelli et al, Diabetes 51:347-355 (2002), Trostchansky et al,Free Radic. Biol. Med. 35:1293-1300 (2003), Mackensen et al, J.Neurosci. 21:4582-4592 (2001), Aslan et al, Proc. Natl. Acad. Sci. USA98:15215-15220 (2001), Sheng et al, Free Radic. Biol. Med. Inpreparation (2003)). Based on the structure-activity relationship, thatrevealed the key roles of metal-centered redox potential(Batinic-Haberle et al, Inorg. Chem. 38:4011-4022 (1999)) andelectrostatics (Spasojevic et al, J. Biol. Chem. 278:6831-6837 (2003))on the superoxide (O₂.⁻) dismuting ability, a similar compound,N,N′-diethylimidazolyl derivative, MnTDE-2-ImP⁵⁺, was synthesized andwas proven effective in vivo (Sheng et al, Drug News and Perspectives15:654-665 (2002), Sheng et al, Free Radic. Biol. Med. 33:947-961(2002), Sheng et al, Free Radic. Biol. Med. In preparation (2003),Bottino et al, Diabetes 51:2561-2567 (2002), Bowler et al, Free Radic.Biol. Med. 33:1141-1152 (2002)). Besides dismuting O₂.⁻, Mn(III) orthoN-alkylpyridylporphyrins are able to efficiently scavenge peroxynitrite(k>10⁷M⁻¹ s⁻) (Ferrer-Sueta et al, J. Biol. Chem. 278:27432-27438(2003), Ferrer-Sueta et al, Chem. Res. Toxicol. 12:442-449 (1999)) andcarbonate radical (k>10⁸ M⁻¹ s⁻¹) (Ferrer-Sueta et al, J. Biol. Chem.278:27432-27438 (2003)). In addition, these Mn porphyrins undergoreductive nitrosylation with NO. (Spasojevic et al, Nitric Oxide:Biology and Chemistry 4:526-533 (2000)). Finally, they are readilyreduced by cellular reductants such as ascorbic acid (Batinic-Haberle etal, J. Chem. Soc., Dalton Trans., pgs. 2689-2696 (2002), Ferrer-Sueta atal, Chem. Res. Toxicol. 12:442-449 (1999), Spasojevic et al, NitricOxide: Biology and Chemistry 4:526-533 (2000), Bloodsworth et al, FreeRadic. Biol. Med. 28:1017-1029 (2000)), glutathione (Spasojevic et al,Nitric Oxide: Biology and Chemistry 4:526-533 (2000)),tetrahydrobiopterin (Spasojevic and Fridovich, Free Radic. Biol. Med.33(Suppl. 2):S316 (2002)), and uric acid (Trostchansky et al, FreeRadic. Biol. Med. 35:1293-1300 (2003), Ferrer-Sueta et al, Chem. Res.Toxicol. 12:442-449 (1999)). Thus, the catalytic elimination of O₂.⁻,ONOO⁻, and CO₃.⁻ by Mn porphyrins is likely made possible in vivothrough coupling with cellular reductants. Through modulation of thelevels of reactive oxygen (ROS) and nitrogen (RNS) species, Mnporphyrins can favorably affect cellular redox status and redoxsensitive signaling processes (Mikkelsen and Wardman, Oncogene22:5734-5754 (2003), Chen et al, Free Radic. Biol. Med. 35:117-132(2003)).

In vivo studies (Trostchansky et al, Free Radic. Biol. Med. 35:1293-1300(2003), Sheng et al, Free Radic. Biol. Med. In preparation (2003))indicated that the efficacy of Mn porphyrins can be improved byincreasing their lipophilicity. Hence, a series of orthoN-alkylpyridylporphyrins were prepared, wherein the length of N-pyridylalkyls was increased from methyl to n-octyl (Batinic-Haberle et al, J.Chem. Soc., Dalton Trans., pgs. 2689-2696 (2002)). While all Mnporphyrins of the series can scavenge O₂.⁻ (Batinic-Haberle et al, J.Chem. Soc., Dalton Trans., pgs. 2689-2696 (2002)) and ONOO⁻(Ferrer-Sueta et al, J. Biol. Chem. 278:27432-27438 (2003)) with nearlyequal effectiveness, their in vivo performance differs greatly. Anincrease in the length of the alkyl chains increases lipophilicity up to10-fold (Batinic-Haberle et al, J. Chem. Soc., Dalton Trans., pgs.2689-2696 (2002)). Consequently, their bioavailibility can be expectedto increase as well. However, as the alkyl chains lengthen, thesurfactant character of the porphyrin increases, leading to thepotential for increased toxicity. At higher concentrations this effectmay predominate over any gain in activity resulting from increasedbioavailibility. An attempt was made to overcome the toxicity by workingat low concentrations where the sole impact of lipophilicity would beassessed.

The present invention results from studies involving modification of theortho N-alkylpyridyl and di-ortho N,N′-dialkylimidazolyl chains byintroducing ether oxygen. 2-Methoxyethyl analogues of MnTE-2-PyP⁵⁺ (FIG.1, MnTMOE-2-PyP⁵⁺) and of MnTDE-2-ImP⁵⁺ (FIG. 1, MnTDMOE-2-ImP⁵⁺) weresynthesized. When compared to ortho pyridylporphyrins, di-orthoimidazolyl compounds have both imidazolyl nitrogens substituted withethyl(methyl) or methoxyethyl groups. The ortho N-alkylpyridylporphyrinsexist as a mixture of positional (atropo-) isomers (Spasojevic et al,Inorg. Chem. 41:5874-5881 (2002)), whereas di-ortho imidazolyl compoundswith eight identical imidazolyl substituents do not have positionalisomers. As described in the Example that follows, the potency andtoxicity of the new Mn porphyrins were assessed using the SOD-deficientE. coli model of oxidative stress (Batinic-Haberle et al, J. Biol. Chem.273:24521-24528 (1998), Batinic-Haberle et al, Inorg. Chem. 38:4011-4022(1999)) which has been proven useful in the past in evaluatingprospective candidates for animal models of oxidative stress injuries.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Structures of the Mn(III) porphyrins studied.

FIG. 2. ESMS of metal-free porphyrins obtained in water:acetonitrile=1:1at 20 V cone voltage.

FIG. 3. ESMS of Mn(III) porphyrins obtained in water:acetonitrile=1:1 at20 V cone voltage.

FIG. 4. Log k_(cat) vs μ_(1/2)/(1+μ^(1/2)) for Mn^(III)TE-2-PyP⁵⁺,Mn^(III)TnBu-2-PyP⁵⁺, MnTMOE-2-PyP⁵⁺ and Mn^(III)TDMOE-2-ImP⁵⁺ obtainedin 0.05 M phosphate buffer, pH 7.8, 0-0.4 M NaCl. Slopes are given inparentheses.

FIGS. 5A-5D. Aerobic growth curves of wild type (FIG. 5A, 5B) andSOD-deficient E. coli (FIG. 5C, 5D) in minimal 5AA minimal medium in theabsence (1) and presence of 3 μM (FIG. 5A, 5C), and 25 μM Mn(III)porphyrins (FIG. 5B, 5D). Mn porphyrins are abbreviated as follows:MnT(alkyl)-2-PyP⁵⁺, alkyl being methyl (2), ethyl (3), n-propyl (4),n-butyl (5), n-hexyl (6), n-octyl (7), MnTMOE-2-PyP⁵⁺ (8), MnTDE-2-ImP⁵⁺(9), MnTM,MOE-2-PyP⁵⁺ (10), MnTDMOE-2-ImP⁵⁺ (11). Inset: Aerobic growthof SOD-deficient E. coli in 5AA minimal medium (A_(700 nm)) after 14hours in the presence of 3 μM (FIG. 5C) and 25 μM (FIG. 5D) Mnporphyrins.

FIGS. 6A-6D. Aerobic growth curves of SOD-deficient E. coli in M9CAmedium in the absence (1) and presence of 1 μM (FIG. 6A), 3 μM (FIG.6B), 10 μM (FIG. 6C) and 30 μM (FIG. 6D) Mn porphyrins. Mn porphyrinsare abbreviated as in FIG. 5. Inset: Aerobic growth of SOD-deficient E.coli in M9CA medium after 6 hours (A_(700 nm)) in the absence (1) andpresence of 1 μM (FIG. 6A), 3 μM (FIG. 6B), 10 μM (FIG. 6C) and 30 μM(FIG. 6D) Mn porphyrins.

FIGS. 7A-7D, Aerobic growth curves of wild type (FIG. 7A, 7C) andSOD-deficient E. coli (FIG. 7B, 7D) in M9CA medium in the presence of 0,0.1, 0.3, 1, 3, 10 and 30 μM MnTnHex-2-PyP⁵⁺ (nHex) (FIG. 7A, 7B) andMnTnOct-2-PyP⁵⁺ (nOct) (FIG. 7C, 7D). Inset: Aerobic growth of E. coliin M9CA medium after 6 hours (A_(700 nm) in the presence of 0, 0.1, 0.3,1, 3, 10 and 30 μM MnTnHex-2-PyP⁵⁺ (nHex) (FIG. 7A, 7B) andMnTnOct-2-PyP⁵⁺ (nOct) (FIG. 7C, 7D).

FIG. 8. Reactivity of manganese complexes expressed in terms of logk_(cat) vs metal-centered redox potential, E_(1/2). Only water-solubleMn(III) porphyrins are given in the left, linear section of the curvethat obeys Marcus equation (Marcus, Annu. Rev, Phys. Chem. 15:155(1964), Jordan, Reaction Mechanisms of Inorganic and OrganometallicSystems, 2^(nd) Ed., Oxford University Press, New York, (1998), Bennet,Prog. Inorg. Chem. 18:1-176 (1973)) and data are from Batinic-Haberle etal, Inorg. Chem. 38:4011-4022 (1999): (1) MnTCPP³⁻, (2) MnT(TMAP)⁵⁺, (3)MnT(2,6-F₂-3-SO₃—P)P³⁻, (4) MnT(TFTMAP)P⁵⁺, (5) MnT(2,6-Cl₂-3-SO₃—P)P³⁻,(6) MnBM-2-PyP³⁺, (7) MnTM-3-PyP⁵⁺, (8) MnTM-4-PyP³⁵⁺, (9) MnTr-2-PyP⁴⁺,(10) MnTM-2-PyP⁵⁺, (11) MnTE-2-PyP⁵⁺. Data for EUK-8 and MnCl₂ are fromBatinic-Haberle et al, Inorg. Chem. 40:726 (2001), data for Mn(II)cyclic polyamine M40403 from Aston et al, Inorg. Chem. 40:1779 (2001).Data for SOD are from (Ellerby et al, J. Am. Chem. Soc. 118:6556 (1996),Vance and Miller, J. Am. Chem. Soc. 120:461 (1998), Klug-Roth et al, J.Am. Chem. Soc. 95:2786 (1973). Data for Mn^(III)Cl₁₋₄MnTE-4-PyP⁵⁺(12-15) are from Kachadourian et al, Inorg. Chem. 38:391-396 (1999),data for Mn^(II)Br₈MnTM-4-PyP⁴⁺ (16) are from Batinic-Haberle et al,Arch. Biochem. Biophys. 343:225 (1997), and for Mn^(II)Cl₅MnTE-2-PyP⁴⁺(17) from Kachadourian et al, Free Radic. Biol. Med. 25 (Suppl. 1):S17(1998) (triangles). Data for MnTnBu-2-PyP⁵⁺ (a) are from Batinic-Haberleet al, J. Chem. Soc., Dalton Trans., pgs. 2689-2696 (2002), and forMnTMOE-2-PyP⁵⁺ (b), MnTD(II)E-2-ImP⁵⁺ (c,d), MnTM,MOE-2-PyP⁵⁺ (e), andMnTDMOE-2-ImP⁵⁺ (f) are from the present work (squares).

FIG. 9. Aerobic growth of SOD-deficient E. coli in M9CA medium after 6hours, measured as A_(700 nm), in the presence of 1 μM Mn(III)N-alkylpyridylporphyrins as a function of their lipophilicity, R_(f)(Table 1). Alkyl is methyl (M) ethyl (E), n-propyl (nPr), n-butyl (nBu),n-hexyl (nHex) and n-octyl (nOct). Inset: The log k_(cat) of Mn(III)N-alkylpyridylporphyrins as a function of their lipophilicity, R_(f).

FIG. 10. Aerobic growth of SOD-deficient E. coli in 5AA minimal mediumat 14 hours, measured as A_(700 nm), in the presence of 3 μM Mn(III)methoxyethylpyridyl- and imidazolyl porphyrins.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a compound of Formula:

When the compound is of Formula I-VI, each R is, independently,—(CH₂)_(m)CH₂OX or —(CH₂CH₂O)_(n)X,

wherein

-   -   m is 1-6, preferably 1-4, more preferably 1 or 2;    -   n is 3-50, preferably 3-10, more preferably 3, 4 or 5;    -   X is C₁₋₁₂alkyl (straight chain or branched), preferably        C₁₋₈alkyl, more preferably C₁₋₄alkyl. Each R can be the same.

When the compound is of Formula VII or VIII, at least one R on eachimadazole ring is, independently, —(CH₂)_(m)CH₂OX or —(CH₂CH₂O)_(n)X,the other R being, independently, a C₁-C₁₂alkyl, (straight chain orbranched), preferably a C₁₋₈alkyl, more preferably a C₁, C₂, C₃ or C₄alkyl;

wherein

-   -   m is 1-6, preferably 1-4, more preferably 1 or 2;    -   n is 3-50, preferably 3-10, more preferably 3, 4 or 5;    -   X is C₁₋₁₂alkyl (straight chain or branched), preferably        C₁₋₈alkyl, more preferably C₁₋₄alkyl. Advantageously, each R is        the same and is —(CH₂)_(n)CH₂OX.

When the compound is any of Formulas I-VIII, each A is, independently,hydrogen or an electron withdrawing group, for example, a halogen (e.g.,Cl, Br or F), NO₂, or CHO, preferably each A is hydrogen or halogen,more preferably at least one A is halogen and the remaining A's arehydrogen, still more preferably 1-4 A's are, independently, Cl or Br andthe remaining A's are hydrogen. M is metal selected from the groupconsisting of manganese, iron, copper, cobalt, nickel and zinc(preferably manganese). Z⁻ is a counterion (e.g., chloride).

To the extent that Hunt et al, Chem. Biol. 4:846-858 (1997), Szabó etal, Mol. Med. 8:571-580 (2002), and/or Mabley et al, Mol. Med. 8:581-590(2002) may describe compounds within the scope of the definitions setforth above, those specific compounds are outside the scope of thecompound per se and/or method embodiments of the present invention.

The invention further relates to a method of protecting cells (egmammalian cells) from oxidant-induced toxicity comprising contacting thecells with a protective amount of a compound as described above. Theinvention further relates to a method of treating a pathologicalcondition of a patient resulting from oxidant-induced toxicitycomprising administering to the patient an effective amount of such acompound. The invention also relates to a method of treating apathological condition of a patient resulting from degradation of NO.,comprising administering to the patient an effective amount of acompound as described above. Additionally, the invention relates to amethod of treating a patient for inflammatory lung disease comprisingadministration to the patient an effective amount of a compound asdescribed above. The inflammatory lung disease can be a hyper-reactiveairway disease. The disease can be asthma.

Additionally the invention relates to a method of treating aneurodegenerative condition of a patient comprising administering to thepatient an effective amount of a compound as described above.Neurodegenerative disease can be familial amyotrophic lateral sclerosis,Parkinson disease, Picks disease, Alzheimers, spinal cord injury,stroke, multiple sclerosis, Mad cow disease, Jacob-Creutzfeld disease.

Additionally the invention relates to a method of treating radiationinjury and cancer of a patient comprising administering to the patientan effective amount of a compound as described above.

Additionally the invention relates to a method of treating a diabeticcondition of a patient comprising administering to the patient aneffective amount of a compound as described above.

Additionally the invention relates to a method of treating a cardiaccondition of a patient comprising administering to the patient aneffective amount of a compound as described above.

Additionally the invention relates to a method of treating a sickle celldisease condition of a patient comprising administering to the patientan effective amount of a compound as described above.

The compounds disclosed herein can be used in the treatment of thediseases, disorders and conditions described in U.S. Pat. No. 5,994,339,U.S. Pat. No. 6,127,356, U.S. Pat. No. 6,479,477, WO 99/23097, U.S. Pat.No. 6,544,975, WO 01/96345, WO 02/060383 and PCT/US02/17144.

The compounds described above, metal bound and metal free forms, can beformulated into pharmaceutical compositions suitable for use in thepresent methods. Such compositions include the compound(s) (activeagent) together with a pharmaceutically acceptable carrier, excipient ordiluent. The composition can be present in dosage unit form, forexample, tablets, capsules or suppositories. The composition can also bein the form of a sterile solution suitable for injection ornebulization. Compositions can also be in a form suitable for opthalmicuse. The invention also includes compositions formulated for topicaladministration, such compositions taking the form, for example, of alotion, cream, gel or ointment. The concentration of active agent to beincluded in the composition can be selected based on the nature of theagent, the dosage regimen and the result sought.

The dosage of the composition of the invention to be administered can bedetermined without undue experimentation and will be dependent uponvarious factors including the nature of the active agent (includingwhether metal bound or metal free), the route of administration, thepatient, and the result sought to be achieved. A suitable dosage to beadministered IV or topically can be expected to be in the range of about0.01 to 50 mg/kg/day, preferably, 0.1 to 10 mg/kg/day. For aerosoladministration, it is expected that doses will be in the range of 0.001to 5.0 mg/kg/day, preferably, 0.01 to 1 mg/kg/day. Suitable doses willvary, for example, with the compound and with the result sought.

Certain aspects of the present invention will be described in greaterdetail in the non-limiting Example that follow.

Example Experimental Details

General.

MnCl₂×4 H₂O, and Baker-flex silica gel IB TLC plates were purchased fromJ. T. Baker. N,N-dimethylformamide, 2-propanol (99.5+%), NH₄ PF₆(99.99%), NaCl, sodium L-ascorbate, and tetrabutylammonium chloride werefrom Aldrich, while xanthine, K₂CO₃, albumin, bovine (# C-3131) andequine ferricytochrome c (lot #7752) were from Sigma. The ethyl(n-butyl)p-toluenesulfonate and methoxyethyl p-toluenesulfonate were from TCIAmerica. Acetone, ethyl ether (anhydrous), chloroform, NaCl, KOH,KH₂PO₄, methanol, EDTA and KNO₃ were from Mallinckrodt and acetonitrilewas from Fisher Scientific. Tris (ultra pure) was from ICN Biomedicals,Inc. Xanthine oxidase was prepared by R. Wiley and was supplied by K. V.Rajagopalan (Waud et al, Arch. Biochem. Biophys. 19:695-701 (1975)).Catalase was from Boehringer, and ultrapure argon from National WeldersSupply Co.

Mn(III) Porphyrins.

Synthesis.

The H₂T-2-PyP, H₂™-2-ImP and H₂T-2-ImP were supplied from MidCenturyChemicals (Chicago, Ill.). The N-ethylation, N-butylation andN-methoxyethylation of H₂T-2-PyP and N-methoxyethylation of H₂™-2-ImPwere accomplished as previously described (Batinic-Haberle, MethodsEnzymol. 349:223-233 (2001), Batinic-Haberle et al, J. Biol. Chem.273:24521-24528 (1998)). However, the method proved unsuccessful whenN,N′-dimethoxyethylation of H₂T-2-ImP was attempted to prepareH₂TDMOE-2-ImP⁴⁺. Thus, no single fully quaternized product was obtained.In this case, N,N′-methoxyethylation was conducted underalkaline/anhydrous conditions to facilitate the proton release (Milgromet al, Tegrahedron 52:9877-9890 (1996)). Thus, 30 mg of H₂T-2-ImP in 7mL of DMF was purged with argon, then 300 mg of K₂CO₃ was added followedby the addition of 7 mL of methoxyethyl p-toluene sulfonate (higheramount of K₂CO₃ will lead to the formation of undesired products). Thequaternization was followed by thin-layer chromatography on silica gelplates with KNO₃satH₂O/H₂O/acetonitrile=1/1/8. The isolation of thechloride salt was performed as previously described (Batinic-Haberle etal, J. Biol. Chem. 273:24521-24528 (1998), Batinic-Haberle et al, Inorg.Chem. 38:4011-4022 (1999)). As was the case with longerN-alkylpyridylporphyrins, diethyl ether alone instead of diethylether/2-propanol, 1/1, was used to rinse the PF₆ ⁻ salt. The sameconditions were then applied for N,N′-diethylation of H₂T-2-ImP.Insertion of manganese into the porphyrin ligand was achieved aspreviously described for N-alkylpyridylporphyrins. The formation ofMn(II) porphyrin occurs readily. The oxidation of manganese was mostrapid with N-methoxyethylpyridylporphyrin and the slowest (within anhour) with N,N′-dimethoxyethylimidazolylporphyrin. The purification andisolation of Mn complexes was the same as with their respective ligands.Elemental analysis: H₂TMOE-2-PyPCl₄×9 H₂O(C₅₂H₇₂N₈O₁₃Cl₄). Found: C,53.93; H, 6.09; N, 10.05; Cl, 12.73. Calculated: C, 53.89; H, 6.26; N,9.67; Cl, 12.24. MnTMOE-2-PyPCl₅×9 H₂O (C₅₂H₇₀N₈O₁₃MnCl₅). Found: C,49.97; H, 4.83; N, 10.32; Cl, 14.77. Calculated: C, 50.07; H, 5.65; N,8.98; Cl, 14.21. H₂TM,MOE-2-ImPCl₄×12.5 H₂O×1.5 NH₄Cl(C₄₈H₈₉N₁₂O_(16.5)Cl_(5.5)). Found: C, 43.70; H, 6.25; N, 13.99; Cl,14.74. Calculated: C, 43.87; H, 6.82; N, 14.38; Cl, 14.84.MnTM,MOE-2-ImPCl₅×12.5 H₂O×0.5 NH₄Cl (C₄₈H₈₁N₁₂O_(16.5)MnCl_(5.5)).Found: C, 42.64; H, 5.70; N, 13.07; Cl, 14.11. Calculated: C, 42.73; H,6.19; N, 12.98; Cl, 14.45. H₂TDMOE-2-PyPCl₄×8 H₂O (C₅₆H₉₀N₁₂O₂₄Cl₄).Found: C, 50.73; H, 6.56; N, 12.74; Cl, 11.73. Calculated: C, 50.60; H,6.82; N, 12.64; Cl, 10.67. MnTDMOE-2-PyPCl₅×12.5H₂O(C₅₆H₉₇N₁₂O_(20.5)MnCl₅). Found: C, 44.78; H, 6.18; N, 11.40; Cl,11.95. Calculated: C, 44.88; H, 6.52; N, 11.21; Cl, 11.82.H₂TDE-2-ImPCl₄×8.5 H₂O(C₄₈H₇₅N₁₂O_(8.5)Cl₄). Found: C, 52.57; H, 6.86;N, 15.27; Cl, 12.66. Calculated: C, 52.50; H, 6.88; N, 15.31; Cl, 12.91.MnTDE-2-ImPCl₅×14 H₂O(C₄₈H₈₄N₁₂O₁₄Cl₅Mn). Found: C, 44.78; H, 6.51; N,13.11; Cl, 13.60. Calculated: C, 44.85; H, 6.58; N, 13.08; Cl, 13.79.

Uv/vis spectra of porphyrin ligands and their Mn complexes were taken ona Shimadzu UV-2501-PC spectrophotometer at 25° C. Thin-layerchromatography of ligands and Mn complexes was done using silica gelplates with KNO₃satH₂O/H₂O/acetonitrile=1/1/8.

Electrochemistry.

Measurements were performed on a CH Instruments Model 600 VoltammetricAnalyzer (Spasojevic and Batinic-Haberle, Inorg. Chim, Acta. 317:230-242(2001), Batinic-Haberle et al, J. Biol. Chem. 273:24521-24528 (1998),Batinic-Haberle et al, Inorg. Chem. 38:4011-4022 (1999)). Athree-electrode system in a small volume cell (0.5 mL to 3 mL), with a 3mm-diameter glassy carbon button working electrode (BioanalyticalSystems), plus the Ag/AgCl reference and Pt auxiliary electrodes wasused. Argon-purged solutions contained 0.05 M phosphate buffer, pH 7.8,0.1 M NaCl, and 0.5 mM metalloporphyrin. The scan rates were 0.01-0.5V/s, typically 0.1 V/s. The potentials were standardized against thepotassium ferrocyanide/ferricyanide (Kolthof and Tomsicek, J. Phys.Chem. 39:945 (1935)) and/or against MnTE-2-PyP⁵⁺ (Spasojevic andBatinic-Haberle, Inorg. Chim. Acta. 317:230-242 (2001), Batinic-Haberleet al, J. Biol. Chem. 273:24521-24528 (1998), Batinic-Haberle et al,Inorg. Chem. 38:4011-4022 (1999)).

Electrospray Mass Spectrometry.

ESMS measurements were performed on a Micromass Quattro LC triplequadrupole mass spectrometer equipped with a pneumatically assistedelectrostatic ion source operating at atmospheric pressure as previouslydescribed (Batinic-Haberle et al, J. Porphyrins Phthalocyanines4:217-227 (2000)). Typically, the 0.5 mM 50% aqueous acetonitrilesolutions of chloride salts of metal-free porphyrins or their Mn(III)complexes were introduced by loop injection into a stream of 50% aqueousacetonitrile flowing at 8 μL/min. Mass spectra were acquired incontinuum mode, scanning from 100-500 m/z in 5 s, with cone voltages of20 V and 30 V. The mass scale was calibrated using polyethylene glycol.

Catalysis of O₂.⁻ Dismutation.

The catalytic rate constants for the O₂.⁻ dismutation were determined bypulse radiolysis in 0.05 M phosphate buffer (pH 7.8) at (22±1)° C. aspreviously described (Batinic-Haberle et al, Inorg. Chem. 40:726(2001)). In the present study 10 mM formate was used as a scavenger forH. atoms and .OH radicals, which leads first to production of CO₂.⁻radicals and then to O₂.⁻ radicals. This system is known to lead toconversion of all primary radicals of water radiolysis into O₂.⁻.

The pulse radiolysis was used as an alternative method to cytochrome cassay in order to circumvent problems encountered with differentcytochrome c preparations. Namely, the k_(cat) values of each porphyrin,determined by cytochrome c assay (McCord and Fridovich, J. Biol. Chem.244:6049 (1969)), varied from 0.1 to 0.5 log units depending upon theparticular cytochrome c preparation used. Sigma-Fluka product #2327009(#30396) previously used is no longer available (Batinic-Haberle et al,Anal. Biochem. 275:267 (1999)). Problems experienced with Sigmacytochrome c #2506 for studying positively charged Mn porphyrins havebeen reported (Batinic-Haberle et al, Anal. Biochem. 275:267 (1999)).Similar problems were encountered with cytochrome c #3131. Incomparative studies, however, the cytochrome c assay was still usedbecause of its simplicity and product #7752 has been found the best forthis purpose among those presently offered by Sigma.

Kinetic Salt Effect.

The dependence of the catalytic rate constant for the O₂.⁻ dismutationupon ionic strength was determined by cytochrome c assay (McCord andFridovich, J. Biol. Chem. 244:6049 (1969)) in 0.05 M phosphate buffer,pH 7.8 with NaCl ranging from 0 to 0.4 M. In doing this, the effect ofionic strength on the rate of the O₂.⁻/cytochrome c reaction wascorrected for.

Aerobic Growth of E. coli.

Bacterial strains of E. coli used were wild type AB1157 (F-thr-1 leu-1proA2 his-4 argE3 thr-1 lacY1 galK2 rpsL supE44 ara-14 xyl-15 mtl-1tsx-33) and SOD-deficient JI132 (same as AB1157 plus (sodk:mudPR13)25(sodB-kan)1-Δ2. Culture media were prepared as follows. M9 medium: 6 g/LNa₂HPO₄, 3 g/L KH₂PO₄, 0.5 g/L NaCl, 1 g/L NH₄Cl, 2 mM MgSO₄, 0.1 mMCaCl₂, adjusted to pH 7.4 with NaOH, and supplemented with 0.2% glucose,3 mg/L each of D-panthotenic acid and thiamine. M9CA medium contained M9medium plus 0.2% casamino acids. 5-amino acid (5AA) minimal mediumcontained M9 medium plus 0.5 mM each of threonine, leucine, histidine,proline and arginine (Faulkner et al, J. Biocl. Chem. 269:23471 (1994)).

AB1157 and 31132 were precultured overnight aerobically in M9CA mediumat 37° C. and were then diluted to 3×10⁷ cells/mL into 5 mL M9CA medium..JI132 was precultured in the presence of 20 μg/mL chloramphenicol and500 μg/mL kanamycin. When the cells are transferred from M9CA medium to5AA minimal medium cultures were first centrifuged and washed twice withthe 5AA medium before final dilution in it. The centrifugation wasconducted at room temperature to avoid temperature shocks, Cultureswithout and with Mn porphyrins (0.1 to 30 μM) were grown aerobically in5 mL volume in test tubes in a thermostated orbital shaker (45° angle,200 rpm) at 37° C.). Rates of growth were followed turbidimetrically at700 nm to minimize interference from the absorbance of test compounds.

Catalysis of O₂.⁻ Dismutation in the Presence of E. coli Cell Extractand Albumin.

The cytochrome c assay (McCord and Fridovich, J. Biol. Chem. 244:6049(1969)) was used to test SOD-like activity in the presence and absenceof E. coli cell extract. SOD-deficient E. coli (JI132, ΔsodA/ΔsodB) wasused for cell extract preparations. Extracts were prepared from 6hour-cultures which were washed twice in 0.05 M phosphate buffer,re-suspended in the buffer and disrupted with a French press. The lysatewas clarified by centrifugation and the supernatant used forexperiments. The SOD activity was examined with or without 31132 celllysate. Before examination, the cell extracts containing 1, 10, or 100μg/mL proteins were incubated for 4 hours at 4° C. with Mn porphyrins.

Results

Thin-Layer Chromatography.

The retention factors, R_(f) (porphyrin path/solvent path) forporphyrins ligands and their Mn complexes are given in Table 1. All newmethoxyporphyrins are more lipophilic than MnTE-2-PyP⁵⁺ andMnTDE-2-ImP⁵⁺.

Uv/Vis Spectroscopy.

The porphyrins obeyed the Beer-Lambert law from 10⁻⁷ M to 10⁻⁵ M, andthe uv/vis data are given in Table 2. A red shift averaging 1-2 nm wasgenerally observed as the result of the increased porphyrin nucleusdistortion due to the enhanced crowding upon replacement of n-butyl withmethoxyethyl groups. The red shift was accompanied by an increase in gof up to 0.1 log unit. The Soret bands of di-ortho H₂TDE-2-ImP⁴⁺ and itsMn complex are blue-shifted by 7 and 8 nm, respectively when compared tothe mono ortho pyridyl analogues as a consequence of increasedelectron-withdrawing effect due to charge delocalization. Thisobservation parallels the markedly increased metal-centered redoxpotential of imidazolylporphyrins vs pyridylporphyrins.

In the presence of ascorbic acid, at pH 7.8 (0.05 M tris buffer), underaerobic conditions, Mn(III) porphyrins are readily reduced. Under theseconditions porphyrins underwent oxidative degradation. As previouslyobserved (Batinic-Haberle et al, J. Chem. Soc., Dalton Trans., pgs.2689-2696 (2002)), those porphyrins that are of more positive E_(1/2)are stabilized in +2 oxidation state and are less prone to oxidativedamage. Accordingly, 27% and 24% of MnTE-2-PyP⁵⁺ and MnTMOE-2-PyP⁵⁺undergo degradation within 2 hours, but only 2% of MnTM,MOE-2-ImP⁵⁺ andMnTDMOE-2-ImP⁵⁺ and 3% of MnTDE-2-ImP⁵⁺. The same level of stability waspreviously observed with n-hexyl and n-octyl porphyrins (Batinic-Haberleet al, J. Chem. Soc., Dalton Trans., pgs. 2689-2696 (2002)).

Electrochemistry.

All cyclic voltammograms, ascribed to the Mn(III)/Mn(II) redox couple,are reversible. The metal-centered redox potentials, E_(1/2) are listedin Table 1. The E_(1/2) of di-ortho ethylimidazolylporphyrin,MnTDE-2-ImP⁵⁺ is 118 mV more positive than of orthoethylpyridylporphyrin, MnTE-2-PyP⁵⁺ (+228 mV vs NHE), as a consequenceof charge delocalization. A similar difference of 114 mV was seenbetween their methoxyethyl analogues. Mn porphyrins bearing N-pyridylsubstituents of the same length, MnTnBu-PyP⁵⁺ and MnTMOE-2-PyP⁵⁺, havenearly identical E_(1/2) of +254 and +251 mV vs NHE, which is 26 and 23mV higher than that of MnTE-PyP⁵⁺.

Electrospray Mass Spectrometry.

ESMS has proven invaluable in identifying metal-free porphyrins andtheir Mn complexes (Batinic-Haberle et al, J. Chem. Soc., Dalton Trans.,pgs. 2689-2696 (2002), Batinic-Haberle et al; J. PorphyrinsPhthalocyanines 4:217-227 (2000)). When done at low cone voltage of 20V, the spectra clearly reflect solvation and ion pairing, redoxproperties, protonation/deprotonation pattern, and dealkylation of thesecompounds.

Metal-Free Porphyrins.

ESMS at 20 V Cone Voltage.

The ESMS spectra are shown in FIG. 2 and the assignments of peaks inTable 3. The ESMS of metal-free porphyrins showed dominant ions assignedto H₂P⁴⁺/4 and/or its monodeprotonated analogue, H₂P⁴⁺—H⁺/3. As is thecase with H₂TnBu-2-PyP⁴⁺, the major peak in ESMS of its methoxyethylanalogue, H₂TMOE-2-PyP⁴⁺, is the monodeprotonated species. Nearly equalabundances of molecular and monodeprotonated ions were observed withH₂TM,MOE-2-ImP⁴⁺ and H₂TDMOE-2-ImP⁴⁺. Negligible double deprotonationwas observed in the case of H₂TMOE-2-PyP⁴⁺ and minor peaks were notedwith H₂TM,MOE-2-ImP⁴ and H₂TDMOE-2-ImP⁴⁺. Only minor solvation wasobserved with H₂TE-2-PyP⁴⁺ and H₂TMOE-2-PyP⁴⁺.

Methoxyethyl porphyrins are more prone to fragmentation when compared totheir alkylated analogues. Loss of ethyl or methoxyethyl groups is lessfrequent with di-ortho substituted imidazolyl porphyrins. This may bedue to the stabilization of these porphyrins by ethyl(methyl) ormethoxyethyl chains distributed both above and below the porphyrinplane. The most stable towards fragmentation is H₂TDE-2-ImP⁴⁺, whichundergoes negligible loss of an ethyl group (2.5% abundance) in contrastto H₂TE-2-PyP⁴⁺ where 25% of monodeethylated species was seen. No lossof methyl groups were observed in the ESMS of H₂TM,MOE-2-ImP⁴⁺.

In addition to the loss of methoxyethyl groups, species consistent withthe loss of .CH₂OCH₃ were observed. These can only result from thefission within N⁺—CH₂—CH₂—O—CH₃. Similar findings were previouslyreported for mesoporphyrin-IX dimethyl ester, where the so-called“benzylic” fission of .CH₂—COO—CH₃ from methyl propionate was detected(Smith, Mass Spectrometry of Porphyrins and Metalloporphyrins inPorphyrins and Metalloporphyrins, Smith, ed., Elsevier ScientificPublishing Company, Amsterdam, p. 388 (1975)).

ESMS at 30 V Cone Voltage.

At higher cone voltage the ratio of monodeprotonated to molecular ionincreases. Also higher abundances of species that result from the lossof multiple N-pyridyl substituents were seen. Again, the most stablecompound was H₂TDE-2-ImP⁴⁺ where no loss of two ethyl groups wasobserved. With H₂TDMOE-2-ImP⁴ only negligible loss of two methoxyethylgroups were observed.

Mn(III) Porphyrins.

ESMS at 20 V Cone Voltage.

The ESMS spectra are shown in FIG. 3, and the assignments of peaks aregiven in Table 3. Species solvated with 1 to 5 acetonitriles wereobserved. The negligible solvation of parent ligands argues for thelocation of solvent molecules around the metal site. The least solvatedis the most lipophilic MnTnBu-2-PyP⁵⁺.

Molecular ions were detected at low abundance. All Mn porphyrins studiedundergo facile reduction. Thus, in ESMS spectra, the major peaks thatare of similar intensities, relate to the oxidized and reduced Mnporphyrins associated with one or two chlorines. The longer theN-pyridyl and N,N′-di-imidazolyl substituents the higher abundance ofspecies associated with two chlorines. In addition, due to hydrogenbonding, higher abundances of chlorinated ions were seen in the ESMS ofmethoxyethyl compounds when compared to their alkyl analogues. Theabundances of oxidized and reduced species were nearly identical in eachof the ESMS other than in the case of MnTDMOE-2-ImP⁵⁺, where more of theoxidized species was seen. This observation is surprising in view of itshigh E_(1/2), but is consistent with it showing the lowest k_(cat) amongthe methoxyethyl compounds. In the spectra of all methoxyethyl compoundsdoubly reduced species were seen. Similar observations with n-butyl,n-hexyl and n-octyl porphyrins were previously reported, and such peakswere assigned to species that are doubly reduced at the metal site(Mn^(I)P³⁺/3) or at both the metal site and porphyrin ring(Mn^(II)P³⁺./3).

Mn(III) pyridyl porphyrins that bear n-butyl or methoxyethyl groups ofthe same overall chain length have very similar patterns offragmentation. In their ESMS major peaks that are of similar abundances,relate to the monochlorinated reduced porphyrin and monochlorinatedspecies that lost one N-pyridyl substituent. MnTMOE-2-PyP⁵⁺ is morehydrophilic, permitting increased hydrogen bonding inside the cavity.Therefore, more solvated species, species that lost charged N-pyridylsubstituent/s and the species associated with two chlorines wereobserved.

As was the case with the metal-free ligands, Mn methoxyethyl porphyrinsare more prone to fragmentation than their alkyl analogues. As with theligands, Mn di-ortho imidazolyl porphyrins become stabilized towardsfragmentation. Namely, only minor loss of methoxyethyl (MnTDMOE-2-ImP⁵⁺)or ethyl (MnTDE-2-ImP⁵⁺) groups was seen. The same has previously beenobserved with long-chain N-alkylpyridylporphyrins such as the n-hexyland n-octyl compounds (Batinic-Haberle et al, J. Chem. Soc., DaltonTrans., pgs. 2689-2696 (2002)). Thus, the abundance of monodealkylatedspecies was increased from methyl to n-butyl, and decreased from n-butylto n-octyl (Batinic-Haberle et al, J. Chem. Soc., Dalton Trans., pgs.2689-2696 (2002)). As with the ligands, fission within N⁺—CH₂—CH₂—O—CH₃was observed with loss of the .CH₂—O—CH₃ radical (Smith, MassSpectrometry of Porphyrins and Metalloporphyrins in Porphyrins andMetalloporphyrins, Smith, ed., Elsevier Scientific Publishing Company,Amsterdam, p. 388 (1975)).

ESMS at 30 V Cone Voltage.

At the higher cone voltage, solvation was only observed withMnTDMOE-2-ImP⁵⁺ suggesting that it has a large capacity for hydrogenbonding due to the highest overall number of oxygen atoms. At highercone voltage the abundance of oxidized species is greatly decreased. Themajor ions correspond to the monochlorinated reduced species except inthe case of MnTDMOE-2-ImP⁵⁺, where the abundance of reduced species hasbeen increased by a mere 5% when compared to the ESMS obtained at 20 V.More of the monodealkylated and monodemethoxylated species were observedat higher cone voltage except in the ESMS of MnTDMOE-2-ImP⁵⁺. Nosignificant differences in fragmentation at 20 V and 30 V were observedfor MnTDMOE-2-ImP⁵⁺.

Catalysis of O₂.⁻ Dismutation.

Pulse radiolysis was used to assess the SOD-like activities of the Mnporphyrins studied. The initial concentration of O₂.⁻ produced by thepulse was about 27 μM, and the concentration of the Mn porphyrin wasbetween 0.5 and 5 μM. The dismutation of O₂.⁻ was followed at 280 nm.The catalytic rate constant was determined from the linear dependence ofk_(obs) upon the concentration of the catalyst (Batinic-Haberle et al,Inorg. Chem. 40:726 (2001)). The log k_(cat) values are summarized inTable 1. All the new compounds have high O₂.⁻ dismuting ability. Themagnitude of the catalysis is due to the interplay of theelectron-withdrawing effects of quaternized ortho pyridyl or di-orthoimidazolyl groups, favorable electrostatics, and the solvation of themetal site.

With N-alkylpyridylporphyrins, it was previously observed that increasedlipophilicity, i.e. desolvation of the porphyrinic compounds, led toincreased E_(1/2). This is because the more deshielded positiveN-pyridyl charges impose a stronger electron-withdrawing effect.Therefore, MnTnBu-2-PyP⁵⁺ has higher E_(1/2) value than MnTE-2-PyP⁵⁺,but is catalytically less potent (Table 1). Such data had beenpreviously explained by the interplay of solvation andelectrostatic/steric effects (Batinic-Haberle et al, J. Chem. Soc.,Dalton Trans., pgs. 2689-2696 (2002)). In this study, evidence arose insupport of a dominant solvation effect and is discussed below. Namely,methoxyethylpyridylporphyrin was synthesized that has N-pyridylsubstituents of the same size that impose similar steric effects as don-butyl side chains in MnTnBu-2-PyP⁵⁺. In addition, MnTMOE-2-PyP⁵⁺ andMnTnBu-2-PyP⁵⁺ have the same E_(1/2), but MnTMOE-2-PyP⁵⁺ has 6-foldhigher k_(cat) (Table 1). The methoxyethyl compound is more hydratedmostly due to the promotion of hydrogen bonding induced by the presenceof oxygen in place of the —CH₂ group in contrast to the lipophilicn-butyl porphyrin. Therefore, the differences in local dielectricconstant alone may be responsible for the higher k_(cat) value obtainedin the case of MnTMOE-2-PyP⁵⁺. Such an observation agrees well with theexplanation offered by Ferrer-Sueta et al for the reaction of Mn(III)N-alkylpyridylporphyrins with ONOO⁻ (Ferrer-Sueta et al, J. Biol. Chem.,278:27432-27438 (2003)). Those authors observed equal sensitivity ofpK_(a,ax) (acid dissociation constant of the axially bound water), andk(ONOO⁻) to the changes in the length of the alkyl chains, as reportedhere with k_(cat). Since the proton dissociation of an axial water is aunimolecular process, such observations can only be ascribed to changesin the local dielectric constant (Batinic-Haberle et al, J. Chem. Soc.,Dalton Trans., pgs. 2689-2696 (2002)). That presumably accounts also forthe observed sensitivity of metallation and demetallation rates ofMn(III) N-alkylpyridylporphyrins to the changes in the length of alkylchains (Espenson, Chemical Kinetics and Reaction Mechanisms, McGraw-HillBook Company, New York, p. 172 (1981)).

Kinetic Salt Effect.

The effect of the ionic strength (μ) on the catalytic rate constant wasassessed using eq [1] which is based on Debye-Huckel relation (Hambrightet al, J. Porphyrins Phthalocyanines 7:139-146 (2003)) for the effect ofthe ionic strength of the solution on the activity coefficient of anion.

log k=log k _(ref)+2Az _(A) z _(B)μ^(1/2)/(1+μ^(1/2))  [1]

The k is the rate constant at any given ionic strength, while k_(ref) isthe rate constant at μ=0. The A is a collection of physical constantswith a value of 0.509 and z_(A) and z_(B) are the charges of thereacting species. The equation predicts a linear plot of log k vsμ^(1/2)/(1+μ^(1/2)). Eq [1] assumes a coefficient of 1.0 (βα_(i)) forμ^(1/2) in the denominator, i.e., the distance of the closest approach,α_(i) to be 3 A and β is a physical constant, 0.33×10⁻¹⁰ m⁻¹. It isdoubtful whether great significance can be attributed to the α_(i), thusto the product z_(A)z_(B) (Hambright et al, J. PorphyrinsPhthalocyanines 7:139-146 (2003)), especially so in the light of thebulkiness, high charge and solvation shell of the metalloporphyrins.Accounting for the mono- and diprotonated phosphates as the majorspecies at pH 7.8 (pK_(a)=7.2), and the concentration of the NaCl, theionic strength was calculated using equation μ=½Σm_(i)z_(i) ² wherem_(i) is the molality and z_(i) the charge of the given ion.

Linear plots of log k_(cat) vs μ^(1/2)/(1+μ^(1/2)) (eq [1]) arepresented in FIG. 4. The slopes of the plots are −6.48 (MnTE-2-PyP⁵⁺),−7.02 (MnTMOE-2-PyP⁵⁺), −7.38 (MnTDMOE-2-ImP⁵⁺), and −4.15(MnTnBu-2-PyP⁵⁺). As expected (Hambright et al, J. PorphyrinsPhthalocyanines 7:139-146 (2003)), when the reactants are ions ofopposite charges, the higher the ionic strength of the solution thelower the rate constants, i.e. the slopes are negative. The slopes ofthe plots indicate clearly the impact of the local dielectric constanton the k_(cat). The k_(cat) of the most lipophilic compound,MnTnBu-2-PyP⁵⁺ is 40% less sensitive to the changes in ionic strength ofthe solution than is the k_(cat) of its methoxyethyl analogue,MnTMOE-2-PyP⁵⁺.

Aerobic Growth of E. coli.

The effects of the Mn porphyrin mimics of SOD activity on the aerobicgrowth of SOD-deficient and wild type E. coli were examined in both the5AA minimal and M9CA media, under aerobic conditions.

5AA Minimal Medium.

FIGS. 5A and 5B show that the growth of the wild type E. coli in 5AAminimal medium was not significantly influenced by these compounds up to25 μM with the exception of the n-hexyl and n-octyl porphyrins whichwere toxic. Thus at 3 μM n-hexyl slowed the growth (line 6, FIG. 5A)while the n-octyl porphyrin completely inhibited growth of the wild typeE. coli (line, FIG. 5A). In contrast, the SOD-deficient strain could notgrow in the 5AA minimal medium (lines 1 in 5C and 5D), and the SODmimics facilitated growth. Thus the n-hexyl porphyrin was most effectiveat 3 μM (line 6 in FIG. 5C), and the n-butyl analogue was nearly aseffective (line 5 in FIG. 5C). The MnTDMOE-2-PyP⁵⁺ (line 11 in FIG. 5C)was the most effective methoxyethyl porphyrin, and was more protectivethan MnTE-2-PyP⁵⁺ (line 3, FIG. 5C) and MnTDE-2-ImP⁵⁺ (line 9, FIG. 5C).When tested at 25 μM the MnTE-2-PyP⁵⁺ and its n-propyl analogue weremost effective (lines 3 and 4, FIG. 5D); with methyl,methoxyethylpyridyl and dimethoxyethylimidazolyl porphyrins not farbehind (FIG. 5D, lines 2, 5, 8 and 11). The n-hexyl porphyrin that hadcomplemented at 3 μM (line 6 in FIG. 5C) failed to do so at 25 μM;presumably due to toxicity at the higher concentration. The insets inFIGS. 5C and 5D allow more rapid assessment of the effects of thesecompounds on the growth of the SOD-deficient E. coli.

M9CA Medium.

Similar to the case in 5AA minimal medium, all of the porphyrins, otherthan longer alkyl chain analogues, were not toxic to wild type E. coli,up to 30 μM levels. Thus, at 30 μM n-hexyl and n-octyl compoundsprevented wild type E. coli to grow, while only marginal toxicity wasobserved with 30 μM n-butyl. Although unable to grow in aerobic minimalmedium, the SOD-deficient strain does grow slowly in M9CA medium. Theeffects of 1, 3, 10 and 30 μM SOD mimics on the growth of theSOD-deficient strain are presented in FIG. 6A-D. The n-hexyl porphyrinwas most effective at 1 and 3 μM (line 6 in FIGS. 6A and 6B), but wasprogressively less effective at 10 and 30 μM (line 6 in FIGS. 6C and6D); undoubtedly due to its toxicity at these high concentrations. Then-butyl analogue, that was most effective at 10 and 30 μM, was lesseffective in promoting growth at 1 and 3 μM (line 5 in FIGS. 6A-D).

MnTM-2-PyP⁵⁺, MnTME-2-PyP⁵⁺, MnTnPr-2-PyP⁵⁺, MnTDE-2-ImP⁵⁺, as well asmethoxyethyl porphyrins facilitated aerobic growth of SOD-deficient E.coli in concentration-dependent manner. At 1 μM all of them (lines 2, 3,4, 8-11, FIG. 6B) offered low protection to SOD-deficient E. coli. At 3μM levels MnTDMOE-2-ImP⁵⁺ (line 11 in FIG. 6B) was the most effective(next to n-butyl and n-hexyl), and was more protective than MnTE-2-PyP⁵⁺(line 3, FIG. 6B), while MnTDE-2-ImP⁵⁺ was ineffective (line 9, FIG.6B). At 30 μM all of them offered either much higher or near fullprotection to SOD-deficient E. coli (lines, 2-4, 8-11, FIG. 6D).

The data in FIGS. 5 and 6 show that the long chain alkyl groups providegreat efficacy that was offset by a concentration-dependent toxicity. Itwas therefore of interest to examine the growth promoting activity ofthe n-hexyl and n-octyl porphyrins over a wide range of concentrations.The effects of these compounds in the range of 0.1 to 30 μM on thegrowth of both SOD-deficient and SOD-proficient strains are presented inFIGS. 7A-D. FIG. 7A shows that the n-hexyl begins to exert toxicity onthe parental strain at 3 μM. FIG. 7C demonstrates the greater toxicityof the octyl compound that becomes observable already at 0.3 μM. Then-butyl compound exerted marginal toxicity at 30 μM. Therefore, itappears that for each increase in the number of carbon atoms by 2(CH₂—CH₂—) from n-butyl to n-octyl, the toxicity increases ˜10-fold.

In accord with these results, the n-hexyl compound was most able tofacilitate the growth of SOD-deficient strain between 0.3 and 3 μM (FIG.7B), while the utility of n-octyl porphyrin was restricted to ≧0.3 μM(FIG. 7D). At 0.3 μM n-hexyl and n-octyl porphyrins (FIGS. 7B and 7D)are equally protective as are 10 μM MnTM-2-PyP⁵⁺, MnTE-2-PyP⁵⁺ andMnTDMOE-2-ImP⁵⁺ (FIG. 6C, lines 2, 3 and 11).

Catalysis of O₂.⁻ Dismutation in the Presence and Absence of E. coliCell Extract.

It has been previously found that ortho MnTM-2-PyP⁵⁺ associates lesswith DNA and RNA as compared to the planar para isomer, MnTM-4-PyP⁵⁺(Batinic-Haberle et al, J. Biol. Chem. 273:24521-24528 (1998)).Consequently, MnTM-4-PyP⁵⁺ was able to catalyze O₂.⁻ dismutation in thepresence of E. coli cell extract only if nucleic acids were removed fromit. Further, Tjahjono et al (Tjahjono et al, Biochim. Biophys. Acta1472:333-343 (1999)) reported that diortho metal-freetetrakis(N,N′-dimethylimidazolium-2-yl)porphyrin interacted with calfthymus DNA ˜10-fold less than H₂TM-4-PyP⁴⁺. In line with these findings,all of the new compounds would not associate with nucleic acids as theyare non-planar, bulky, either ortho pyridyl- ordiorthoimidazolyl-substituted porphyrins. Thus, they exhibited the sameSOD-like activity in the presence and absence of E. coli cell extract.

Discussion

O₂.⁻ Dismuting Ability of Mn Porphyrins.

A linear relationship has been established between the log k_(cat) forthe O₂.⁻ dismutation (eqs [2] and [3])³ and metal-centered redoxpotential of Mn(III)

Mn^(III)P+O₂.⁻<====>Mn^(II)P⁻+O₂ ,k _(red)  [2]

Mn^(II)P⁻+O₂.⁻+2H⁺<====>Mn^(III)P⁻+H₂O₂ ,k _(ox)  [3]

porphyrins (Batinic-Haberle et al, Inorg. Chem. 38:4011-4022 (1999)).The relationship obeys the Marcus equation (Marcus, Annu. Rev. Phys.Chem. 15:155 (1964), Jordan, Reaction Mechanisms of Inorganic andOrganometallic Systems, 2^(nd) Ed., Oxford University Press, New York(1998), Bennet, Prog. Inorg. Chem. 18:1-176 (1973)) whereby k_(cat)increases 10-fold with each 120 mV increase in E_(1/2). The Marcusequation, for outer-sphere one-electron transfer reactions (Marcus,Annu. Rev. Phys. Chem. 15:155 (1964)) is applicable because k_(cat) isdetermined by the rate-limiting reduction of Mn(III) porphyrins withO₂.⁻ (eq [2], see the rising limb of the bell shape curve in FIG. 8).The most potent catalysts which emerge from this structure-activityrelationship, MnTM-2-PyP⁵⁺ and MnTE-2-PyP⁵⁺, owe their potency to theelectron-withdrawing effect of quaternized ortho pyridyl nitrogens.These ortho porphyrins are more bulky than their planar para isomers,and that minimizes the unfavorable interactions with DNA and RNA; avital property if the compounds are to be used as SOD mimics in vivo. At+228 mV vs NHE (1,2,5) the reduction of MnTE-2-PyP⁵⁺ by O₂.⁻ proceedswith a rate constant of 2.5×10⁷ M⁻¹ s¹ and oxidation by 8.2×10⁷ M⁻¹ s⁻¹(1). That is the case because the midway potential (+360 mV vs NHE) forthe reduction and oxidation of O₂.⁻ is being approached, which providesan equal thermodynamic driving force for both half-reactions ofcatalytic cycle (eqs [2] and [3]). Thus, Cu,Zn-SOD with E_(1/2) of ˜+300mV vs NHE reduces and oxidizes O₂.⁻ with equal rate constants of 2×10⁹M⁻¹ s⁻¹ (pH 7.8) (Vance and Miller, Biochemistry 40:13079 (2001), (a)Lawrence and Sawyer, Biochemistry 18:3045 (1979), (b) Barrette et al,Biochemistry 22:624 (1983), Ellerby et al, J. Am. Chem. Soc. 118:6556(1996), Vance and Miller, J. Am. Chem. Soc. 120:461 (1998), Klug-Roth etal, J. Am. Chem. Soc. 95:2786 (1973)). Any further increase or decreasein E_(1/2) stabilizes Mn+2 or +3 oxidation state so that eitheroxidation of Mn(II) compounds (M40403 (Cuzzocrea et al, Br. J.Pharmacol. 132:19-29 (2001), Aston et al, Inorg. Chem. 40:1779 (2001),Riley, Adv. Supramol. Chem. 6:217-244 (2000), (a) Riley et al, Inorg.Chem. 35:5213 (1996), (b), Riley and Weiss, J. Am. Chem. Soc. 116:387(1994)) and Mn^(II)Cl₂ (Batinic-Haberle et al, Inorg. Chem. 40:726(2001)) or reduction of Mn(III) compounds, respectively, becomerate-limiting step accompanied by a decrease in log k_(cat). The Marcusequation is again obeyed in the region where the oxidation of Mn(II)compounds is a rate-limiting step (falling limb of FIG. 8). While theSOD-like activity of Mn porphyrins (Batinic-Haberle et al, Inorg. Chem.38:4011-4022 (1999), Kachadourian et al, Inorg. Chem. 38:391-396 (1999),Batinic-Haberle et al, J. Chem. Soc., Dalton Trans., pgs. 2689-2696(2002), Batinic-Haberle et al, Arch. Biochem. Biophys. 343:225 (1997),Kachadourian et al, Free Radic. Biol. Med. 25(Suppl 1):S17 (1998)) hasbeen maximized thermodynamically, kinetic enhancement remains possible.

Study of the structural basis for the antioxidant ability ofMnTE-2-PyP⁵⁺ led to the design of the imidazolyl analogue,MnTDE-2-ImP⁵⁺. Both compounds have been proven beneficial in differentmodels of oxidative stress injuries (Tao et al, Circulation108:2805-2811 (2003), Sheng et al, J. Neurotrauma, In press (2003),Sheng et al, Drug News and Perspectives 15:654-665 (2002), Sheng et al,Free Radic. Biol. Med. 33:947-961 (2002), Vujaskovic et al, Free Radic.Biol. Med. 33:857-863 (2002), Piganelli et al, Diabetes 51:347-355(2002), Trostchansky et al, Free Radic. Biol. Med. 35:1293-1300 (2003),Mackensen et al, J. Neurosci. 21:4582-4592 (2001), Aslan et al, Proc.Natl. Acad. Sci. USA 98:15215-15220 (2001), Sheng et al, Free Radic.Biol. Med. In preparation (2003), Bottino et al, Diabetes 51:2561-2567(2002), Bowler et al, Free Radic. Biol. Med. 33:1141-1152 (2002)). TheMnTDE-2-ImP⁵⁺ has both ortho imidazolyl nitrogens substituted with alkylchains. The positive charge is thus delocalized over both nitrogens,providing greater proximity to the meso porphyrin carbons, which imposesa stronger electron-withdrawing effect than does the positively chargedortho pyridyl nitrogens. Consequently, MnTDE-2-ImP⁵⁺ and otherimidazolyl compounds synthesized in this work have more than 100 mVhigher E_(1/2) than analogous pyridyl porphyrins (Table 1). However, forthe reasons discussed above, no further increase in k_(cat) has beengained.

It has been shown that both k_(cat)(O₂.⁻) (Batinic-Haberle et al, J.Biol. Chem. 273:24521-24528 (1998), Batinic-Haberle et al, Inorg. Chem.38:4011-4022 (1999), Batinic-Haberle et al, J. Chem. Soc., DaltonTrans., pgs. 2689-2696 (2002)) and the rate constant for ONOO⁻(Ferrer-Sueta et al, J. Biol. Chem. 278:27432-27438 (2003), Ferrer-Suetaet al, Chem. Res. Toxicol. 12:442-449 (1999)) reduction by Mn porphyrinsare proportional to the electron-deficiency of the porphyrin (measuredas E_(1/2) for Mn^(III)/Mn^(II) redox couple), and are thus proportionalto each other (Ferrer-Sueta et al, J. Biol. Chem. 278:27432-27438(2003)). It can therefore be expected that these new compounds will beeffective scavengers of ONOO⁻ as well. (Although the reduction of ONOO⁻involves the O═Mn^(IV)/Mn^(III) redox couple, the dependence of the rateconstant for ONOO⁻ reduction by Mn(III) porphyrins upon the E_(1/2) ofMn^(III)/Mn^(II) redox couple is observed because the rate-limiting stepin reduction of ONOO⁻ is its binding to the manganese (Ferrer-Sueta etal, J. Biol. Chem. 278:27432-27438 (2003)). Further, as theirreducibility is increased along with increased E_(1/2), they would bemore readily reduced by cellular reductants, whereby the pro-oxidantaction of Mn(III) or O═Mn(IV) porphyrins would be prevented(Batinic-Haberle et al, J. Chem. Soc., Dalton Trans., pgs. 2689-2696(2002), Trostchansky et al, Free Radic. Biol. Med. 35:1293-1300 (2003),Ferrer-Sueta et al, Chem. Res. Toxicol. 12:442-449 (1999), Bloodsworthet al, Free Radic. Biol. Med. 28:1017-1029 (2000)). As alreadymentioned, Mn porphyrins have the ability to scavenge a wide range ofROS and RNS. That may make them advantageous over more selectiveantioxidants such as cyclic polyamines (Cuzzocrea et al, Br. J.Pharmacol. 132:19-29 (2001), Aston et al, Inorg. Chem. 40:1779 (2001),Riley, Adv. Supramol. Chem. 6:217-244 (2000), (a) Riley et al, Inorg.Chem. 35:5213 (1996), (b), Riley and Weiss, J. Am. Chem. Soc. 116:387(1994)), that were reported to lack reactivity towards H₂O₂, ONOO⁻, NOand HClO (Cuzzocrea et al, Br. J. Pharmacol. 132:19-29 (2001)).

Improving Bioavailability of Mn Porphyrins.

Stroke (Sheng et al, Free Radic. Biol. Med. 33:947-961 (2002)) andspinal cord injury (Sheng et al, Free Radic. Biol. Med. In preparation(2003)) studies indicate that the toxicity and bioavailability ofMnTE-2-PyP⁵⁺ and MnTDE-2-ImP⁵⁺ could be favorably modified. Theimidazolyl compound exerts 16-fold lower neurotoxicity in a stroke modelthan does the pyridyl compound (Sheng et al, Drug News and Perspectives15:654-665 (2002), Sheng et al, Free Radic. Biol. Med. 33:947-961(2002), Mackensen et al; J. Neurosci. 21:4582-4592 (2001)). Since bothcompounds are of similar in vitro antioxidant potency, the lowerneurotoxicity of MnTDE-2-ImP⁵⁺ was ascribed to its higher metal-centeredredox potential and/or bulkiness as it has ethyl groups distributedabove and below the porphyrin plane. However, possibly for the samereasons, MnTDE-2-ImP⁵⁺ lacks protectiveness in spinal cord injury ascompared to MnTE-2-PyP⁵⁺ when both compounds are given intravenously(Sheng et al, Free Radic. Biol. Med. In preparation (2003)). Inaddition, the excessive hydrophilic character of both compounds limitstheir transport across the blood brain and spinal cord barrier and thusdiminishes their ability to protect (Sheng et al, Free Radic. Biol. Med.33:947-961 (2002)).

The lipophilicity and bulkiness of the parent compound, MnTE-2-PyP⁵⁺,has been increased by increasing the length of N-pyridyl alkyl chains upto n-octyl (Batinic-Haberle et al, J. Chem. Soc., Dalton Trans., pgs.2689-2696 (2002)). With no loss of antioxidant capacity thelipophilicity was increased nearly 10-fold from methyl to n-octylcompound (Batinic-Haberle et al, J. Chem. Soc., Dalton Trans., pgs.2689-2696 (2002)), Table 1 and FIG. 9A, inset). The E. coli study showsthat the increase in the alkyl chain length increases the toxicity ofthese compounds, so that 30 μM n-hexyl and n-octyl porphyrins preventwild type E. coli from growing. In a separate experiment both metal-freen-octyl and its Mn(III) complex proved equally toxic. Thus, it may bethe surfactant character, rather than the redox property, of theporphyrin that causes the toxicity. However, at 100-fold lowerconcentration (0.3 μM), the toxicity of n-hexyl and n-octyl compoundswas mostly eliminated, and at 0.3 μM both compounds offered the samelevel of protection as the methyl and ethyl porphyrins did at 10 μM, andthe n-butyl did at 3 μM.

FIG. 9 indicates a similarity by which in vitro SOD potency (k_(cat)) ofN-alkylpyridylporphyrins and protection of SOD-deficient E. coli dependupon lipophilicity index, R_(f). From methyl to n-propyl the protectionof SOD-deficient E. coli decreases slightly as a consequence of thedecrease in k_(cat) (FIG. 6B, lines 2, 3 and 4). The decrease in k_(cat)with increase in R_(f) occurs because the ionic dismutation reactionsare hindered by the local lipophilicity (FIG. 9A, inset). (From methylto n-octyl metal-centered redox potential increases linearly with R_(f)(Batinic-Haberle et al, J. Chem. Soc., Dalton Trans., pgs. 2689-2696(2002)). As the lipophilicity increases further from n-butyl to n-octyl,the effect of the metal-centered redox potential prevails and k_(cat)starts to increase (FIG. 9A, inset). Eventually, methyl and n-octyl areof same SOD-like potency (discussed in detail in Batinic-Haberle et al,J. Chem. Soc., Dalton Trans., pgs. 2689-2696 (2002)). The ability toprotect E. coli follows the same trend; yet, rather than from n-butyl,it increases from n-propyl to n-octyl (lines 5 and 6 in FIG. 6B, FIG.9). More importantly, the increase in protection increases at higherpace than does SOD-like activity. Therefore, a 30-fold higher protectionof n-octyl vs methyl porphyrin is probably due to ˜10-fold higherlipophilicity of n-octyl compound.

A new series of analogues of potent SOD mimics, MnTE-2-PyP⁵⁺ andMnTDE-2-ImP⁵⁺ have been described herein (FIG. 1). MnTMOE-2-PyP⁵⁺ is amethoxyethyl analogue of MnTE-2-PyP⁵⁺ with two-fold higher SOD-likeactivity. It is similarly bulky as MnTnBu-2-PyP⁵⁺ since it has one —CH₂group of each n-butyl chain replaced with oxygen. Moreover, suchmodification diminishes surfactant character of the alkyl chains. TheMnTMOE-2-PyP⁵⁺ has the same metal-centered redox potential of +251 mV vsNHE as the n-butyl compound (+254 mV vs NHE), but a 6-fold highercatalytic potency (Table 1), the highest among the Mn porphyrin-basedSOD mimics thus far prepared. The enhanced antioxidant capacity whencompared to the MnTnBu-2-PyP⁵⁺ is due to the increased hydration ofMnTMOE-2-PyP⁵⁺ that favors ionic O₂.⁻ dismutation reactions (eqs [2] and[3]). Such a conclusion is also supported by a much greater sensitivityof the k_(cat) of MnTMOE-2-PyP⁵⁺ to the ionic strength variations (FIG.4).

In the same manner, the imidazolyl compound, MnTDE-2-ImP⁵⁺ has beenmodified. Two porphyrins were synthesized that have either all eightethyl groups replaced by methoxyethyl chains (MnTDMOE-2-ImP⁵⁺), or havethe imidazole nitrogens substituted by four methyl and four methoxyethylgroups (MnTM,MOE-2-ImP⁵⁺) (FIG. 1).

All three newly synthesized methoxyethylporphyrins have similar SOD-likeactivity and lipophilicity (Table 1), and are not toxic to E. coli underall conditions tested. Their k_(cat) values are the same in the presenceand absence of E. coli cell extract. However, the new compounds differin their ability to protect E. coli from oxidative stress. At ≦3 μMlevels MnTDMOE-2-ImP⁵⁺ with highest number of oxygen atoms is the mostprotective to E. coli among methoxyethyl porphyrins (FIG. 10). It isalso more protective than MnTE-2-PyP⁵⁺, while MnTDE-2-ImP⁵⁺ is the leasteffective. While MnTDMOE-2-ImP⁵⁺ and MnDTE-2-ImP⁵⁺ have very similark_(cat), E_(1/2) and R_(f) (Table 1), the latter lacks oxygens in itsstructure, and that may be the source of its inefficiency. The data arein accord with a spinal cord study which suggests that MnTDE-2-ImP^(s+)was less effective in passing the spinal cord blood barrier thanMnTE-2-PyP⁵⁺, when both compounds are given intravenously (Sheng et al,Free Radic. Biol. Med. In preparation (2003)).

In summary, it has been shown that the modification of the catalyticantioxidants MnTE-2-PyP⁵⁺ and MnTDE-2-ImP⁵⁺, either by increasing thelength of the side chains or introducing oxygen atoms, results incompounds, such as MnTnHex(nOct)-2-PyP⁵⁺ and MnTDMOE-2-ImP⁵⁺, which canbe promising for the treatment of oxidative stress injuries.

The abbreviations used herein are: Mn^(III/II)P^(5+/4+) any Mn porphyrinin oxidized and reduced state, MnT(alkyl)-2-PyP⁵⁺, Mn(III)5,10,15,20-tetrakis(N-alkylpyridinium-2-yl)porphyrin; alkyl being methyl(M), ethyl (E), n-propyl (nPr), n-butyl (nBu), n-hexyl (nHex) andn-octyl (nOct). MnTDE-2-ImP⁵⁺, Mn(III)5,10,15,20-tetrakis[N,N′-diethylimidazolium-2-yl]porphyrin, thisporphyrin has previously (Sheng et al, Drug News and Perspectives15:654-665 (2002)) and elsewhere) been erroneously abbreviated asMnTDE-1,3-ImP⁵⁺, whereby 1 and 3 were indicating the imidazolylnitrogens, rather than the position 2 where the imidazolyl is attachedto the porphyrin ring; MnTMOE-2-PyP⁵⁺ (MOE), Mn(III) tetrakis5,10,15,20-tetrakis[N-(2-methoxyethyl)pyridinium-2-yl]porphyrin;MnTM,MOE-2-ImP⁵⁺ (M,MOE), Mn(III) tetrakis5,10,15,20-tetrakis[N-methyl-N′-(2-methoxyethyl)imidazolium-2-yl]porphyrin;MnTDMOE-2-ImP⁵⁺ (DMOE), Mn(III) tetrakis5,10,15,20-tetrakis[N,N′-di(2-methoxyethyl)imidazolium-2-yl]porphyrin;NHE, normal hydrogen electrode; SOD, superoxide dismutase; ROS and RNSreactive oxygen and nitrogen species. SOD-deficient, ΔsodA/ΔsodB, JI132E. coli; SOD-proficient wild type, AB1157 E. coli; 5AA, 5 amino acids;M9CA, M9 casamino acids medium; LB, Luria-Bertani Medium; NHE, normalhydrogen electrode. The porphyrin having Mn in its +3 state is assignedas a neutral species.

The entire content of all documents cited herein are incorporated hereinby reference, as is: Improving bioavailability of SOD mimics. Comparisonof new Mn(III) methoxyethylpyridyl- and imidazolylporphyrins withMn(III) N-alkylpyridylporphyrins in complementing SOD-deficient E. coliby Ines Batinic-Haberle, Ayako Okado-Matsumoto, Ivan Spasojevic, RobertD. Stevens, Peter Hambright, Pedatsur Neta, Irwin Fridovich, J. Biol.Chem. Submitted.

Tables

TABLE 1 Metal-centered redox potentials E_(1/2), log k_(cat) for O₂ ⁻dismutation and chromatographic R_(f) values for Mn(III) porphyrins.Porphyrin E_(/12), mV vs NHE^(a) log k_(cat) ^(b) Rf^(c) MnTMOE-2-PyP⁵⁺+251 8.04 0.16(0.18) MnTM, MOE-2-ImP⁵⁺ +356 7.98 0.15(0.17)MnTDMOE-2-ImP⁵⁺ +365 7.59 0.21(0.24) MnTDE-2-ImP⁵⁺ +346 7.83 0.17(0.23)MnTM-2-PyP^(5+ d) +220 7.79 0.09(0.13) MnTE-2-PyP^(5+ d) +228 7.73^(e)0.13(0.21) MnTnPr-2-PyP^(5+ d) +238 7.38 0.20(0.31) MnTnBu-2-PyP^(5+ d)+254 7.25 0.33(0.46) MnTnHex-2-PyP^(5+ d) +314 7.48 0.57(0.63)MnTnOct-2-PyP^(5+ d) +367 7.71 0.08(0.86) ^(a)E_(1/2) (±3 mV) determinedin 0.1M NaCl, 0.05M phosphate buffer, pH 7.8. ^(b)k_(cat) (±30%)determined by pulse radiolysis, pH 7.8, (22 ± 1) ° C.; ^(c)R_(f),compound path/solvent path on silica gel TLC plates in KNO₃-saturatedH₂O/H₂O_(/)acetonitrile = 1/1/8, R_(f) for metal-free porphyrins are inparenthesis. ^(d)Data from Batinic-Haberle et al,, J. Chem. Soc., DaltonTrans., pgs. 2689-2696 (2002). ^(e)Data from Batinic-Haberle et al,Inorg. Chem. 40:726 (2001).

TABLE 2 Molar absorptivities of methoxyethyl porphyrins, H₂TDE-2-ImP⁵⁺and their Mn complexes. Porphyrin λ_(max) (log ε)^(a) H₂TMOE-2-PyP⁴⁺416(5.44), 512.5(4.34), 545(3.68), 584(3.93), 636.5(3.46) H₂TDE-2-ImP⁴⁺407(5.26), 507(4.23), 541(3.83), 578.5 (3.87), 630(3.87) H₂TM,MOE-2-ImP⁴⁺ 408.5(5.27), 507.5(4.26), 541(3.87), 580(3.90), 632.5(3.89)H₂TDMOE-2-ImP⁴⁺ 411(5.22), 509.5(4.19) 542.5(3.82), 581(3.83), 634(3.86)MnTMOE-2-PyP⁵⁺ 364.5(4.75), 413(4.41), 455(5.24), 500(3.84),558.5(4.16), 785(3.34) MnTDE-2-ImP⁵⁺ 348.5(4.66), 446(5.08),505.5(3.77), 553(4.06), 588(3.95), 795(3.34) MnTM, MOE-2-ImP⁵⁺349(4.73), 412.5(4.53), 447.5(5.14), 503.5(3.80), 553.5(4.13),588.5(4.04), 799(3.34) MnTDMOE-2-ImP⁵⁺ 350(4.67), 4.14(4.48),448.5(5.09), 503(3.74), 555(4.08), 590(4.02), 803.5(3.33) ^(a)Molarabsorptivities were determined in water at room temperature.

TABLE 3 ESMS of methoxyethylporphyrins and their alkyl analogues.^(a)m/z Porphyrin E_(PyP) ^(b) nBu^(b) MOE_(PyP) DE_(ImP) M,MOE_(ImP)DMO_(ImP) H₂P⁴⁺/4 184 212 214 201 217 261 H₂P⁴⁺ + AN/4 194 222 224 211227 H₂P⁴⁺-H⁺/3 245 282 285 267 289 347 H₂P⁴⁺-H⁺+ AN/3 258 H₂P⁴⁺-a⁺-H⁺ +CH₃ ⁺/3 270 274 333 H₂P⁴⁺-a⁺/3 235 263 265 258 269 328 H₂P⁴⁺-a⁺-H⁺/2 352394 398 386^(c) 403^(c) 491^(c) H₂P⁴⁺-2a⁺/2 366^(c) 368 372^(c) 374^(c)462^(c) H₂P⁴⁺-2a⁺ + H⁺/3 249^(c) H₂P⁴⁺-a⁺-a-2H⁺ 743^(c) H₂P⁴⁺-3a⁺ 677H₂P⁴⁺-3a⁺ + H⁺/2 239^(c) H₂P⁴⁺-a⁺ + CH₃ ⁺/4 203 206 250 H₂P⁴⁺-2a⁺ + CH₃⁺/3 251^(c) 255 313 H₂P⁴⁺-2a⁺ + 2CH₃ ⁺/4 192 195 239 H₂P⁴⁺-3a⁺ + CH₃ ⁺/2346^(c) 352^(c) H₂P⁴⁺-3a⁺ + 2CH₃ ⁺/3 236 H₂P⁴⁺-3a⁺ + 2CH₃ ⁺-H⁺/2 324^(c)H₂P⁴⁺-4a⁺ + CH₃ ⁺ 633^(c) H₂P⁴⁺-2H⁺/2 367 423 426 400 432 520^(c)H₂P⁴⁺-H⁺ + Cl⁻/2 418 450 ^(a)~0.5 mM solutions of porphyrins in 1:1 =acetonitrile:H₂O, 20 V cone voltage, a is either alkyl or methoxyethylgroup. ^(b)Batinic-Haberle et al,, J. Chem. Soc., Dalton Trans., pgs.2689-2696 (2002). ^(c)30 V cone voltage. The fission of CH₂—O—CH₃radical from methoxyethyl group is noted for simplicity as the loss ofmethoxyethyl (a⁺) and the gain of CH₃ ⁺.

TABLE 4 ESMS of Mn(III) methoxyethylporphyrins, and their alkylanalogues.^(a) m/z Porphyrins E_(PyP) ^(b) nBu^(b) MOE_(PyP) DE_(ImP)M,MOE_(ImP) DMOE_(ImP) Mn^(III)P⁵⁺/5 157 181 171 184 219 Mn^(III)P⁵⁺ +AN/5 166 188 190 179 193 227 Mn^(III)P⁵⁺ + 2AN/5 174 196 198 187 201 236Mn^(III)P⁵⁺ + 3AN/5 182 205 206 195 209 244 Mn^(III)P⁵⁺ + 4AN/5 190 213214 204 252 Mn^(III)P⁵⁺ + 5AN/5 260 Mn^(III)P⁵⁺ + Cl⁻/4 206 234 236 223239 283 Mn^(III)P⁵⁺ + Cl⁻ + AN/4 216 Mn^(III)P⁵⁺ + 2Cl⁻/3 286 323 326309 330 389 Mn^(III)P⁵⁺-a⁺ + Cl⁻/3 265 293 295 357 Mn^(III)P⁵⁺-a⁺/4 212207 215 259 Mn^(III)P⁵⁺-a⁺ + AN/4 200 221 269 Mn^(III)P⁵⁺-2a⁺/3 243 262263 266^(c) 268 Mn^(III)P⁵⁺-2a⁺ + AN/3 276 277 Mn^(III)P⁵⁺-3a⁺/2 365371^(b) Mn^(III)P⁵⁺-3a⁺ + Cl⁻ 765 Mn^(III)P⁵⁺-4a⁺ 671 Mn^(III)P⁵⁺-a⁺ +CH₃ ⁺ + Cl⁻/4 225 272 Mn^(III)P⁵⁺-2a⁺ + CH₃ ⁺ + Cl⁻/3 280Mn^(III)P⁵⁺-4a⁺ + CH₃ ⁺/2 Mn^(III)P⁵⁺-CH₃ ⁺ + H⁺/5 181^(b)Mn^(III)P⁵⁺-Mn³⁺ + H⁺/3 281 Mn^(III)P⁵⁺-Mn³⁺ + 2H⁺/4 214 217Mn^(II)P⁴⁺/4 197 227 214 274 Mn^(II)P⁴⁺ + AN/4 207 235 224 Mn^(II)P⁴⁺ +Cl⁻/3 274 312 314 297 318 377 Mn^(II)P⁴⁺ + 2Cl⁻/2 428 488 462 494 583Mn^(II)P⁴⁺-a⁺/3 253 281 283^(c) 276^(c) 287 Mn^(II)P⁴⁺-2a⁺/2 401^(c)Mn^(II)P⁴⁺-2a⁺ + H⁺/3 326 Mn^(II)P⁴⁺-a⁺ + CH₃ ⁺ + Cl⁻/3 299 304 362Mn^(II)P⁴⁺-4a⁺ + CH₃ ⁺ 687 Mn^(II)P⁴⁺-7a³⁺ 653^(d) Mn^(II)P^(.3+)/3 orMn^(I)P³⁺/3 295 303 285^(c) 307 365 ^(a)~0.5 mM solutions of porphyrinsin 1:1 = acetonitrile:H₂O, 20 V cone voltage, a is either alkyl ormethoxyethyl group. ^(b)Batinic-Haberle et al,, J. Chem. Soc., DaltonTrans., pgs. 2689-2696 (2002). ^(c)30 V cone voltage. ^(d)signal of verylow intensity. The fission of CH₂—O—CH₃ radical from methoxyethyl groupis noted for simplicity as the loss of methoxyethyl (a⁺) and the gain ofCH₃ ⁺.

1. A compound of formula

when the compound is of Formula I-VI, each R is, independently,—(CH₂)_(m)CH₂OX or —(CH₂CH₂O)_(n)X, wherein m is 1-6, n is 3-50, X isC₁₋₁₂alkyl (straight chain or branched), when the compound is of FormulaVII or VIII, at least one R on each imadazole ring is, independently,—(CH₂)_(m)CH₂OX or —(CH₂CH₂O)_(n)X, the other R being, independently, aC₁-C₁₂alkyl, (straight chain or branched), wherein m is 1-6, n is 3-50,X is C₁₋₁₂alkyl (straight chain or branched), when the compound is anyof Formulas each A is, independently, hydrogen or an electronwithdrawing group, M is metal selected from the group consisting ofmanganese, iron, copper, cobalt, nickel and zinc, and Z⁻ is acounterion.
 2. The compound according to claim 1 wherein at least one Ais halogen, NO₂ or CHO.
 3. The compound according to claim 1 whereinsaid compound is of Formula I, III, V or VII and M is manganese
 4. Thecompound according to claim 1 where said compound is of Formula V, VI,VII or VIII.
 5. A method of protecting cells from oxidant-inducedtoxicity comprising treating said cells with a protective amount of thecompound of claim 1 under conditions such that the protection iseffected.
 6. The method according to claim 5 wherein said cells aremammalian cells.
 7. A method of treating a pathological condition of apatient resulting from oxidant-induced toxicity comprising administeringto said patient an effective amount of the compound of claim 1 underconditions such that the treatment is effected.
 8. A method of treatinga pathological condition of a patient resulting from degradation of NO.comprising administering to said patient an effective amount of thecompound of claim 1 under conditions such that the treatment iseffected.
 9. A method of treating a patient for inflammatory lungdisease comprising administering to said patient an effective amount ofthe compound of claim 1 under conditions such that the treatment iseffected.
 10. A method of treating a neurodegenerative condition of apatient comprising administering to the patient an effective amount ofthe compound of claim
 1. 11. A method of treating radiation injury andcancer of a patient comprising administering to the patient an effectiveamount of the compound of claim
 1. 12. A method of treating a diabeticcondition of a patient comprising administering to the patient aneffective amount of the compound of claim
 1. 13. A method of treating acardiac condition of a patient comprising administering to the patientan effective amount of the compound of claim
 1. 14. A method of treatingsickle cell disease in a patient comprising administering to the patientan effective amount of the compound of claim 1.